A simple and effective preparative procedure has been designed and developed for large scale purification of human urinary erythropoietin. This procedure consists of affinity chromatography on Con A-Sepharose 4B and Wheat germ 6MB; hydrophobic chromatogaphy on Phenyl-Sepharose CL4B; gel filtration on Sephacryl S-200 and adsorption chromatography on hydroxylapatite. These steps have been developed as a result of many trials of various combinations of techniques, based on the molecular characteristics and chromatographic behavior of Ep, that we have learned in pilot experiments. This procedure yielded highly purified EP preparation with potencies ranging from 3,500 to 4,000 units/mg of protein. This represented a mean purification factor of 3,125 with an overall recovery of 22 to 35%. The purified material showed a single major component in SDS-PAGE, isoelectric focusing and gel filtration on Sephadex Gl00, which corresponded to a molecular weight of 37,000 to 39,000 daltons, a pI3.7 to 4.0 with a homogeneity of about 90%. In order to remove traces of remaining microheterogenicities and to obtain homogeneous Ep, additional purification steps are being developed, including specific reversed immunoaffinity chromatography.